Chimeric norovirus P particle and preparation and use thereof

ABSTRACT

Provided is a recombinant P particle formed from a norovirus capsid P protein of a chimeric Aβ1-m peptide (m being an integer ranging from 6 to 15), wherein the recombinant P particles form an ordered and repetitive antigen array. Also provided are a nucleotide sequence encoding the recombinant P particle, a pharmaceutical composition comprising same and a use thereof for preparing a medicament for treating or preventing Alzheimer&#39;s disease. Also provided is a method for preparing the recombinant P particle.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application filed under 35U.S.C. § 371 of International Application No. PCT/CN2016/087643, filedJun. 29, 2016, which claims the benefit of Chinese Application No.201510415556.1, filed Jul. 15, 2015. Both of these applications arehereby incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to the fields of molecular biology andimmunology. In particular, the present invention relates to arecombinant P particle formed from a norovirus capsid P protein of achimeric Aβ1-m peptide, wherein the recombinant P particles form anordered and repetitive antigen array, and wherein m is an integerranging from 6 to 15.

BACKGROUND ART

Alzheimer's disease (AD), also named senile dementia, is a progressiveneurodegenerative disease. Its main clinical characteristics includegradual occurrences of memory deterioration, cognitive functiondisorders and abnormal behaviors, and ending up with a loss of theability to live independently. AD leads to death from the complications10-20 years after the onset thereof. Currently, this most commonneurodegenerative disease cannot be cured or effectively delayed, andseverely jeopardizes the physical and mental health and life quality ofthe elderly.

Researchers generally believe that the amyloid-β protein (Aβ) in thebrain tissue is a primary pathogenic cause of neuronal damage andcognitive function disorders. Aβ is derived from its precursor β-amyloidprecursor protein (abbreviated as βAPP). βAPP, as a transmembraneprotein, is ubiquitously present in various tissues of the body andexpressed most highly in the brain tissue. In the amyloid cleavingpathway, βAPP can be cleaved into Aβ proteins which are 38-43 aminoacids in length, wherein Aβ1-42 protein is the main type that formsamyloid deposits.

Aβ active immune vaccine, which is intended to stimulate the body toproduce Aβ antibodies and thus clear up Aβ1-42 deposits in the brain, isan important method for treating Alzheimer's disease. As a pioneer of ADvaccines, Aβ1-42 polypeptide vaccine shows a good therapeutic effect ofdelaying memory decline in the AD transgenic mouse model. Nevertheless,in the clinical trials of Aβ1-42 polypeptide vaccine, 6% of the ADpatients showed the side effect of cephalomeningitis, and they had a lowlevel of Aβ1-42 antibody. The research showed that although Aβ1-42polypeptide vaccine could elicit antibody reactions, it may promotevaccinated patients to produce autoimmune T cell reactions in the brainand thus induce meningoencephalitis as it carries a large number of Tcell epitopes. In comparison with Aβ1-42, polypeptide Aβ1-15 comprisingonly the first 15 amino acids at the N-terminal of Aβ polypeptide,comprises only B cell epitopes instead of T cell epitopes, and thispolypeptide vaccine could stimulate the human body to produce specificimmunological reactions against Aβ1-42 protein and show good safety inhuman trials. Therefore, Aβ1-15 has become an antigenic peptide withgreat research potentials. However, as a hapten, Aβ1-15 does not have agood immunological effect and sustainability of inducing the productionof antibodies. Therefore, there are urgent needs for a vaccine againstAlzheimer's disease with high safety and good immunological effect.

In the prior art, it is often preferred to synthesize the virus-likeparticle and Aβ polypeptide respectively, and then couple them in orderto obtain vaccines with high immunogenicity. However, as to the vaccinesobtained by methods such as coupling, it is difficult to control thenumber of the inserted epitopes, and to purify the vaccines effectively.

SUMMARY OF THE INVENTION

The inventors found that the capsid P protein of norovirus is a veryideal antigen exhibition platform for an Aβ1-m (m is an integer rangingfrom 6 to 15) peptide. When different copies of Aβ1-m (m is an integerranging from 6 to 15) peptide encoding sequences with various lengthsare inserted into the loop of the DNA sequence encoding the capsid Pprotein of norovirus, Aβ1-m can be exhibited on the utmost surface ofthe recombinant P particle formed from the recombinant P protein,thereby helping to stimulate the body to the greatest extent to producespecific immunological reactions against Aβ1-42 proteins. Therecombinant P particle of the present application can be preparedeasily, and are controllable in terms of the number of the insertedepitopes; they can be purified easily and manufactured with a low cost;they have good safety and high immunogenicity, and can induce theproduction of high-titer antibodies.

In the present invention, multiple copies of DNA encoding antigenpeptide Aβ1-m (m is an integer ranging from 6 to 15) are inserted intothe DNA encoding loop1, loop2 and/or loop3 of the P protein by means ofgenetic engineering. By in vitro expression and self-assembly ofrecombinant proteins, a recombinant P particle capable of sufficientlyexhibiting the antigen epitopes of Aβ1-m (m is an integer ranging from 6to 15) is formed, thereby helping to enhance the efficacy,sustainability and safety of vaccination.

In a first aspect, the present invention provides a recombinant Pparticle formed from a norovirus capsid P protein of a chimeric Aβ1-mpeptide, wherein m is an integer ranging from 6 to 15. In an embodimentof the invention, m may be 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. Saidrecombinant P particles form an ordered and repetitive antigen array,wherein the amino acid sequence of at least one of the Aβ1-m peptides isembedded into loop1, loop2 and/or loop3 of the norovirus capsid Pprotein.

In a preferred embodiment of the present invention, the amino acidsequence of a norovirus capsid P protein is shown as SEQ ID NO: 1. Inthe recombinant P particle, N1 Aβ1-m peptide sequences are embeddedbehind one or more amino acid sites selected from the group consistingof amino acids 70-74 of SEQ ID NO:1, i.e. I70, A71, G72, T73 and Q74; N2Aβ1-m peptide sequences are embedded after one or more amino acid sitesselected from the group consisting of amino acids 148-151 of SEQ IDNO:1, i.e. T148, S149, N150 and D151; and N3 Aβ1-m peptide sequences areembedded after one or more amino acid sites selected from the groupconsisting of amino acids 168-171 of SEQ ID NO:1, i.e. D168, G169, S170and T171; wherein N1, N2 and N3 each are independently selected from aninteger ranging from 0 to 40, and N1+N2+N3≥1. In particular, N1, N2 andN3 each are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40, and N1+N2+N3≥1.

That “Aβ1-m peptide sequences are embedded after one amino acid site”means that the N terminal of the Aβ1-m peptide sequences is directlylinked to the C terminal of the amino acid residue via a peptide bond oris linked to the amino acid via an amino acid linker. In a preferredembodiment, the amino acid linker is (Gly)_(n), wherein preferably n is1-10, and more preferably n=3.

The multiple consecutive Aβ1-m peptide sequences inserted into SEQ IDNO:1 can be directly linked or linked via an amino acid linker. In apreferred embodiment, the amino acid linker is (Gly)_(n), whereinpreferably n is 1-10, and more preferably n=3.

In the present invention, an Aβ1-m peptide sequence means thepolypeptide consisting of the first m amino acids at the N terminal ofthe entire Aβ1-42 protein, wherein m is an integer ranging from 6 to 15.For example, the Aβ1-m peptide according to the present invention can bea polypeptide consisting of amino acids 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-13, 1-14 or 1-15 at the N terminal of the entire Aβ1-42 protein.

The Aβ1-m peptide sequence according to the present invention can be anAβ1-m peptide from human, mouse, primate, rabbit, African clawed frog(Xenopuslaevis), rat and guinea pig. The amino acid sequences of Aβ1-15peptides from human, mouse, primate, rabbit, African clawed frog, ratand guinea pig are shown as SEQ ID NO:2 to SEQ ID NO:8, respectively.According to these sequences, those skilled in the art could easilydetermine the Aβ1-m peptide (m is an integer ranging from 6 to 15) ofthe present invention. Preferably, the Aβ1-m peptide is a human Aβ1-mpeptide.

In a second aspect, the present invention further provides a nucleotidesequence encoding a norovirus capsid P protein of a chimeric Aβ1-mpeptide from which the recombinant P particle of the first aspect isformed, wherein m is an integer ranging from 6 to 15. In an embodimentof the invention, m may be 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. Thenucleotide sequence comprises a nucleotide sequence encoding a noroviruscapsid P protein and at least one copy of a nucleotide sequence encodinga Aβ1-m peptide, wherein the at least one copy of a nucleotide sequenceencoding a Aβ1-m peptide is inserted into the nucleotide sequenceencoding a loop of a norovirus capsid P protein, and wherein theinserted nucleotide sequence encoding a Aβ1-m peptide would not resultin a frameshift mutation of the norovirus capsid P protein.

In a specific embodiment, the at least one copy of a nucleotide sequenceencoding a Aβ1-m peptide can be inserted into the nucleotide sequenceencoding the same loop of a norovirus capsid P protein, or into thenucleotide sequences encoding different loops of a norovirus capsid Pprotein. For example, where the nucleotide sequence encoding a noroviruscapsid P protein is SEQ ID NO: 9, loop1 corresponds to nucleotides208-222, loop2 corresponds to nucleotides 442-453, and loop3 correspondsto nucleotides 502-513.

In a specific embodiment of the invention, the numbers of the Aβ1-mpeptide-encoding nucleotide sequences inserted into loop1, loop2 andloop3 are N1, N2 and N3, respectively; N1, N2 and N3 each areindependently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 and 40, and N1+N2+N3≥1.

In a specific embodiment of the invention, the 5′ end nucleotide of thenucleotide sequence encoding a Aβ1-m peptide is linked directly to thenucleotide sequence encoding a loop of a norovirus capsid P protein via3′,5′-phosphodiester bond or via a DNA linker. The insertion would notresult in frame shift during translation of the nucleotide sequence,whether it is a direct insertion or an insertion via a DNA linker.

In a preferred embodiment, the DNA linker is (GGA)n, (GGG)n or (GGC)n,wherein preferably n is 1-10, and more preferably n is 3.

In a specific embodiment of the invention, the multiple consecutiveAβ1-m peptide-encoding nucleotide sequences inserted into the nucleotidesequence encoding a norovirus capsid P protein are linked directly toeach other by 3′,5′-phosphodiester bond or by a DNA linker. Theinsertion would not result in frame shift during translation of thenucleotide sequence, whether it is a direct insertion or an insertion bya DNA linker. In a preferred embodiment, the DNA linker is (GGA)n,(GGG)n or (GGC)n, wherein preferably n is 1-10, and more preferably n is3.

Preferably, the Aβ1-m peptide nucleotide sequence is a nucleotidesequence encoding an Aβ1-m peptide from human, mouse, primate, rabbit,African clawed frog (Xenopuslaevis), rat and guinea pig. Morepreferably, the Aβ1-m peptide nucleotide sequence is a nucleotidesequence encoding an Aβ1-m peptide from human.

In a preferred embodiment of the invention, the nucleotide sequencesencoding norovirus capsid P proteins of chimeric Aβ1-m peptides are SEQID NOs: 15-141. More preferably, the nucleotide sequence encoding anorovirus capsid P protein of a chimeric Aβ1-m peptide is selected fromSEQ ID NO:24, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:57, SEQ ID NO:73,SEQ ID NO:92, SEQ ID NO:117, SEQ ID NO:132, SEQ ID NO:133 and SEQ IDNO:134.

In a third aspect, the present invention also provides a pharmaceuticalcomposition used for preventing or treating Alzheimer's disease,comprising the recombinant P particle in the first aspect of theinvention and a pharmaceutically acceptable carrier. The pharmaceuticalcomposition is preferably used in mammals, and more preferably used inhuman. Preferably, the pharmaceutical composition is a vaccinecomposition, and more preferably, the vaccine composition also comprisesan adjuvant. In a specific embodiment of the invention, the adjuvant isCpG. In another specific embodiment of the invention, the adjuvant is analuminum adjuvant. The vaccine composition is preferably administeredsubcutaneously or nasally, and more preferably administeredsubcutaneously.

In a fourth aspect, the present invention also provides use of therecombinant P particle in the first aspect of the invention in themanufacture of a medicament for treating or preventing Alzheimer'sdisease. Preferably, the medicament is a vaccine. The medicament ispreferably used in mammals, and more preferably used in human.

In a fifth aspect, the present invention also provides a method forpreparing the recombinant P particle in the first aspect of theinvention, comprising the following steps:

i) obtaining an expression vector comprising a nucleic acid encoding anorovirus capsid P protein of a chimeric Aβ1-m peptide, wherein m is aninteger ranging from 6 to 15;

ii) transferring the expression vector into a receptor cell;

iii) expressing the norovirus capsid P protein of a chimeric Aβ1-mpeptide, and allowing it to self-assemble into a recombinant P particle;

and preferably, the method also comprises isolation and purificationsteps.

In a specific embodiment, cation exchange chromatography and/orhydrophobic chromatography can be used for purification.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the maps of the various recombinant pET26b plasmidconstructs.

A is the schematic diagram of pET26b-P protein plasmid that has beenconstructed.

B is the schematic diagram of pET26b-P protein-mEagI plasmid with amEagI restriction enzyme site obtained by mutation.

C is the schematic diagram of pET26b-P protein-mEagI&mKpnI plasmid witha mEagI restriction enzyme site and a mKpnI restriction enzyme siteobtained by mutation.

D is the schematic diagram of pET26b-P protein-10copy-Aβ1-6-loop1G₇₂plasmid with the gene fragment of interest Pprotein-10copy-Aβ1-6-loop1G₇₂ loaded between a mKpnI restriction enzymesite and a SalI restriction enzyme site.

E is the schematic diagram of pET26b-P protein-1copy-Aβ1-6-loop2S₁₄₉plasmid with the gene fragment of interest Pprotein-1copy-Aβ1-6-loop2S₁₄₉ loaded between a mEagI restriction enzymesite and a SalI restriction enzyme site.

F is a schematic diagram of pET26b-Pprotein-10copy-Aβ1-6-loop2S₁₄₉plasmid with the gene fragment of interest Pprotein-10copy-Aβ1-6-loop2S₁₄₉ loaded between a mEagI restriction enzymesite and a SalI restriction enzyme site.

G is the schematic diagram of pET26b-P protein-20copy-Aβ1-6-loop3G₁₆₉plasmid with the gene fragment of interest Pprotein-20copy-Aβ1-6-loop3G₁₆₉ loaded between a mEagI restriction enzymesite and a SalI restriction enzyme site.

H is the schematic diagram ofpET26b-Pprotein-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉ plasmidwith the gene fragment of interestPprotein-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉ loaded between amEagI restriction enzyme site and a mKpnI restriction enzyme site.

I is the schematic diagram of pET26b-Pprotein-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ plasmid with thegene fragment of interestPprotein-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ loaded between amEagI restriction enzyme site and a mKpnI restriction enzyme site.

J is the schematic diagram of pET26b-Pprotein-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉ plasmid with thegene fragment of interest Pprotein-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉ loaded between amEagI restriction enzyme site and a mKpnI restriction enzyme site.

K is the schematic diagram of pET26b-Pprotein-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉plasmid with the gene fragment of interestPprotein-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉loaded between a mEagI restriction enzyme site and a mKpnI restrictionenzyme site.

L is the schematic diagram of pET26b-P protein-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ plasmidwith the gene fragment of interest P protein-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ loadedbetween a mEagI restriction enzyme site and a mKpnI restriction enzymesite.

M is the schematic diagram of pET26b-Pprotein-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉plasmid with the gene fragment of interestPprotein-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉loaded between a mEagI restriction enzyme site and a mKpnI restrictionenzyme site.

FIG. 2 shows the schematic diagrams of the structures of gene fragmentsof interest.

A is the schematic diagram of the locations of three loops of P protein(loop1, loop2 and loop3), together with site-directed mutation sites andrestriction enzyme sites carried by P protein gene;

B is the schematic diagram of a P particle gene fragment of interestwith 10 copies of Aβ1-6 gene embedded behind site G₇₂ into loop1,abbreviated as P protein-10copy-Aβ1-6-loop1G₇₂;

C is the schematic diagram of a P particle gene fragment of interestwith one copy of Aβ1-6 gene embedded behind site S₁₄₉ into loop2,abbreviated as P protein-1copy-Aβ1-6-loop2S₁₄₉;

D is the schematic diagram of a P particle gene fragment of interestwith 10 copies of Aβ1-6 gene embedded behind site S₁₄₉ into loop2,abbreviated as P protein-10copy-Aβ1-6-loop2S₁₄₉;

E is the schematic diagram of a P particle gene fragment of interestwith 20 copies of Aβ1-6 gene embedded behind site G₁₆₉ into loop3,abbreviated as P protein-20copy-Aβ1-6-loop3G₁₆₉;

F is the schematic diagram of a P particle gene fragment of interestwith 10 copies of Aβ1-15 gene embedded behind site G₇₂ into loop1 and 10copies of Aβ1-6 gene embedded behind site S₁₄₉ into loop2, abbreviatedas P protein-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉;

G is the schematic diagram of a P particle gene fragment of interestwith 3 copies of Aβ1-12 gene embedded behind site S₁₄₉ into loop2 and 3copies of Aβ1-6 gene embedded behind site G₁₆₉ into loop3, abbreviatedas P protein-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉;

H is the schematic diagram of a P particle gene fragment of interestwith one copy of Aβ1-6 gene embedded behind site G₇₂ into loop1 and 10copies of Aβ1-6 gene embedded behind site G₁₆₉ into loop3, abbreviatedas P protein-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉;

I is the schematic diagram of a P particle gene fragment of interestwith one copy of Aβ1-6 gene embedded behind site G₇₂ into loop1, onecopy of Aβ1-6 gene embedded behind site S₁₄₉ into loop2 and one copy ofAβ1-6 gene embedded behind site G₁₆₉ into loop3, abbreviated as Pprotein-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉;

J is the schematic diagram of a P particle gene fragment of interestwith 3 copies of Aβ1-6 gene embedded behind site G₇₂ into loop1, 3copies of Aβ1-6 gene embedded behind site S₁₄₉ into loop2 and 3 copiesof Aβ1-6 gene embedded behind site G₁₆₉ into loop3, abbreviated as Pprotein-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉;and

K is the schematic diagram of a P particle gene fragment of interestwith 10 copies of Aβ1-6 gene embedded behind site G₇₂ into loop1, 10copies of Aβ1-6 gene embedded behind site S₁₄₉ into loop2 and 10 copiesof Aβ1-6 gene embedded behind site G₁₆₉ into loop3, abbreviated as Pprotein-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉.

FIG. 3 shows the schematic diagram of 10 recombinant P particles. A isthe schematic diagram of P protein-10copy-Aβ1-6-loop1G₇₂; B is theschematic diagram of Pprotein-1copy-Aβ1-6-loop2S₁₄₉; C is the schematicdiagram of P protein-10copy-Aβ1-6-loop2S₁₄₉; D is the schematic diagramof P protein-20copy-Aβ1-6-loop3G₁₆₉; E is the schematic diagram of Pprotein-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉; F is theschematic diagram of Pprotein-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉; G is the schematicdiagram of P protein-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉; H isthe schematic diagram of Pprotein-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉;I is the schematic diagram of Pprotein-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉;and J is the schematic diagram of Pprotein-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉.

FIG. 4 shows the purification and characterization of recombinant Pparticles. A is the SDS-PAGE and Native-PAGE graphs, as well as theelectron microscope image of PP-10copy-Aβ1-6-loop1G₇₂ protein; B is theSDS-PAGE and Native-PAGE graphs, as well as the electron microscopeimage of PP-1copy-Aβ1-6-loop2S₁₄₉ protein; C is the SDS-PAGE andNative-PAGE graphs, as well as the electron microscope image ofPP-10copy-Aβ1-6-loop2S₁₄₉ protein; D is the SDS-PAGE and Native-PAGEgraphs, as well as the electron microscope image ofPP-20copy-Aβ1-6-loop3G₁₆₉ protein; E is the SDS-PAGE and Native-PAGEgraphs, as well as the electron microscope image ofPP-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉ protein; F is theSDS-PAGE and Native-PAGE graphs, as well as the electron microscopeimage of PP-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ protein; G isthe SDS-PAGE and Native-PAGE graphs, as well as the electron microscopeimage of PP-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉ protein; H isthe SDS-PAGE and Native-PAGE graphs, as well as the electron microscopeimage ofPP-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉protein; I is the SDS-PAGE and Native-PAGE graphs, as well as theelectron microscope image ofPP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉protein; J is the SDS-PAGE and Native-PAGEgraphs, as well as theelectron microscope image ofPP-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉protein.

FIG. 5 shows the determination of optimal immune dosage and optimalimmune adjuvant of PP-10copy-Aβ1-6-loop2S₁₄₉ protein vaccine in aC57BL/6J mouse model.

A is the detection of anti-Aβ42 antibody in mouse immune serum afterimmunization with different dosages of PP-10copy-Aβ1-6-loop2S₁₄₉ proteinvaccine stimulated by different immune adjuvants. B is the detection ofT cell responses produced by mouse spleen lymphocytes after immunizationwith different forms of PP-10copy-Aβ1-6-loop2S₁₄₉ protein vaccine.

FIG. 6 shows the ELISA detection of anti-Aβ42 antibody in mouse immuneserum and the comparison of Aβ42 antibody levels from different immunegroups after the mice are immunized with three different forms ofprotein vaccine. A is the statistical graph of four immunization resultsfor the group of subcutaneous immunization with PBS. B is thestatistical graph of four immunization results for the group of nasalimmunization with PBS. C is the statistical graph of four immunizationresults for the group of subcutaneous immunization with CpG. D is thestatistical graph of four immunization results for the group of nasalimmunization with CpG. E is the statistical graph of four immunizationresults for the group of subcutaneous immunization withPP-1copy-Aβ1-6-loop2S₁₄₉ and the adjuvant CpG. F is the statisticalgraph of four immunization results for the group of nasal immunizationwith PP-1copy-Aβ1-6-loop2S₁₄₉ and the adjuvant CpG. G is the statisticalgraph of four immunization results for the group of subcutaneousimmunization with PP-10copy-Aβ1-6-loop2S₁₄₉ and the adjuvant CpG. H isthe statistical graph of four immunization results for the group ofnasal immunization with PP-10copy-Aβ1-6-loop2S₁₄₉ and the adjuvant CpG.I is the statistical graph of four immunization results for the group ofsubcutaneous immunization withPP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ andthe adjuvant CpG. J is the statistical graph of four immunizationresults for the group of nasal immunization withPP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ andthe adjuvant CpG. K is the comparison of results after the fourthimmunization for each group, wherein the best immunological effect isobserved in the group of subcutaneous immunization withPP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ andthe adjuvant CpG.

SEQUENCE LISTING

SEQ ID NO: 1 is the amino acid sequence of a norovirus capsid P protein.

SEQ ID NOs: 2-8 are amino acid sequences of Aβ1-15 peptides from human,mouse, primate, rabbit, African clawed frog (Xenopuslaevis), rat andguinea pig.

SEQ ID NO: 9 is the nucleotide sequence encoding a norovirus P protein.

SEQ ID NO: 10 is the nucleotide sequence encoding an A1-15 peptide fromhuman.

SEQ ID NO: 11 is a site-directed mutation forward primer used in theprocess of obtaining pET26b-P protein-mEagI plasmid.

SEQ ID NO:12 is a site-directed mutation reverse primer used in theprocess of obtaining pET26b-P protein-mEagI plasmid.

SEQ ID NO: 13 is a site-directed mutation forward primer used in theprocess of obtaining pET26b-P protein-mEagI&mKpnI plasmid.

SEQ ID NO: 14 is a site-directed mutation reverse primer used in theprocess of obtaining pET26b-Pprotein-mEagI&mKpnI plasmid.

SEQ ID NO: 15-SEQ ID NO: 141 are nucleotide sequences encoding noroviruscapsid P proteins of chimeric Aβ1-m peptides prepared in the examples ofthe invention.

SEQ ID NO: 143-SEQ ID NO: 152 are nucleotide sequences of DNA fragmentsof interest synthesized in the process of constructing recombinantplasmids expressing preferred 10 recombinant P proteins, correspondingto 10copy-Aβ1-6-loop1G₇₂, 1copy-Aβ1-6-loop2S₁₄₉, 10copy-Aβ1-6-loop2S₁₄₉,20copy-Aβ1-6-loop3G₁₆₉, 3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉,10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉,1copy-Aβ1-6-loop1-G₇₂-10copy-Aβ1-6-loop3G₁₆₉,1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉,3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉, and10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉,respectively.

DETAILED DESCRIPTION OF THE INVENTION Definition

All the technical terms used in the present invention have the samemeanings as those skilled in the art commonly understand, unlessotherwise indicated.

An ordered and repetitive antigen array refers to multiple Aβ1-mpeptides (m is an integer ranging from 6 to 15) repetitively and orderlyarranged on the surface of a norovirus recombinant P particle.

P protein refers to the P protein in the norovirus capsid P proteins,which can self-assemble in vitro into a P particle. When used herein, Pprotein used at the gene level means a gene fragment, nucleotidesequence, plasmid, etc. encoding a P protein; P protein used at theprotein level means a P protein monomer or dimer. In the accompanyingfigures, all schematic diagrams of protein show P protein, such as theplasmids encoding P proteins shown in FIGS. 1, 2 and 3, and the Pprotein monomer shown in the denatured electrophoresis of FIG. 4.

P particle (abbreviated as PP) refers to a protein particle formed bythe in vitro self-assembly of P protein in norovirus. The most commonform of P particle is a tetracosamer. When used herein, P particle (PP)only used at the protein level means the form of polymer (such as atetracosamer), including various proteins used for property detectionand immunization, such as the polymer form shown in the denaturedelectrophoresis of FIG. 4.

Pprotein-N1copy-Aβ1-m-loop1Ak1-N2copy-Aβ1-m-loop2Ak2-N3copy-Aβ1-m-loop3Ak3 means that N1 copies of Aβ1-m are embedded behind the amino acid atposition K2 into loop1 of P protein, N2 copies of Aβ1-m are embeddedbehind the amino acid at position K2 into loop2 of P protein, and N3copies of Aβ1-m are embedded behind the amino acid at position K3 intoloop3 of P protein, wherein m is an integer ranging from 6 to 15. Unlessotherwise indicated, Aβ1-m is linked to P protein via a polypeptidelinker having three glycines. For example, Pprotein-1copy-Aβ1-6-loop2T₁₄₈-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop2N₁₅₀means a P protein in which one copy of Aβ1-6 is respectively embeddedbehind the amino acid at position 148, behind the amino acid at position149, and behind the amino acid at position 150 into loop2, and eachAβ1-6 is linked to P protein via an amino acid linker having threeglycines encoded by GGA.

P protein-10copy-Aβ1-6-loop2S₁₄₉(GGA)o means that 10 copies of Aβ1-6 areembedded behind the amino acid at position 149 into loop2 of P protein,and there is no linker between Aβ1-6 and P protein, and no amino acidlinker between each Aβ1-6, i.e. Aβ1-6 is directly linked to P protein oranother Aβ1-6 via a peptide bond.

P protein-10copy-Aβ1-6-loop2S₁₄₉(GGC)₃ means that 10 copies of Aβ1-6 areembedded behind the amino acid at position 149 into loop2 of P protein,Aβ1-6 is linked to P protein via an amino acid linker having threeglycines encoded by GGC, and each Aβ1-6 is linked to each other via anamino acid linker having three glycines encoded by GGC.

The technical solutions of the invention are further illustrated by thefollowing examples; however, the present invention is not limited tothese examples. Unless otherwise indicated, the formulations and devicesused herein are all commercially available.

The present invention provides 127 recombinant P particles in whichmultiple copies of Aβ1-m peptides (m is an integer ranging from 6 to 15)are embedded into loop1, loop2 and/or loop3 of P protein. The P proteinsfrom which said 127 recombinant P particles are formed are shown inTable 1.

TABLE 1 The amino acid sequences of the recombinant P proteins fromwhich said 127 recombinant P particles of the present invention areformed and the nucleotide sequences encoding the recombinant P proteins.Nucleotide Sequences encoding the NO. Recombinant P Protein RecombinantP proteins 1 P protein-1copy-Aβ1-6-loop1I₇₀ SEQ ID NO: 15 2 Pprotein-10copy-Aβ1-9-loop1I₇₀ SEQ ID NO: 16 3 Pprotein-20copy-Aβ1-12-loop1I₇₀ SEQ ID NO: 17 4 Pprotein-40copy-Aβ1-15-loop1I₇₀ SEQ ID NO: 17 5 Pprotein-1copy-Aβ1-9-loop1A₇₁ SEQ ID NO: 19 6 Pprotein-10copy-Aβ1-12-loop1A₇₁ SEQ ID NO: 20 7 Pprotein-20copy-Aβ1-15-loop1A₇₁ SEQ ID NO: 21 8 Pprotein-40copy-Aβ1-6-loop1A₇₁ SEQ ID NO: 22 9 Pprotein-1copy-Aβ1-6-loop1G₇₂ SEQ ID NO: 23 10 Pprotein-10copy-Aβ1-6-loop1G₇₂ SEQ ID NO: 24 11 Pprotein-20copy-Aβ1-6-loop1G₇₂ SEQ ID NO: 25 12 Pprotein-40copy-Aβ1-6-loop1G₇₂ SEQ ID NO: 26 13 Pprotein-1copy-Aβ1-12-loop1T₇₃ SEQ ID NO: 27 14 Pprotein-10copy-Aβ1-15-loop1T₇₃ SEQ ID NO: 28 15 Pprotein-20copy-Aβ1-6-loop1T₇₃ SEQ ID NO: 29 16 Pprotein-40copy-Aβ1-9-loop1T₇₃ SEQ ID NO: 30 17 Pprotein-1copy-Aβ1-15-loop1Q₇₄ SEQ ID NO: 31 28 Pprotein-10copy-Aβ1-6-loop1Q₇₄ SEQ ID NO: 32 19 Pprotein-20copy-Aβ1-9-loop1Q₇₄ SEQ ID NO: 33 20 Pprotein-40copy-Aβ1-12-loop1Q₇₄ SEQ ID NO: 34 21 Pprotein-1copy-Aβ1-9-loop2T₁₄₈ SEQ ID NO: 35 22 Pprotein-10copy-Aβ1-9-loop2T₁₄₈ SEQ ID NO: 36 23 Pprotein-20copy-Aβ1-9-loop2T₁₄₈ SEQ ID NO: 37 24 Pprotein-40copy-Aβ1-9-loop2T₁₄₈ SEQ ID NO: 38 25 Pprotein-1copy-Aβ1-6-loop2S₁₄₉ SEQ ID NO: 39 26 Pprotein-10copy-Aβ1-6-loop2S₁₄₉ SEQ ID NO: 40 27 Pprotein-20copy-Aβ1-6-loop2S₁₄₉ SEQ ID NO: 41 28 Pprotein-40copy-Aβ1-6-loop2S₁₄₉ SEQ ID NO: 42 29 Pprotein-1copy-Aβ1-12-loop2N₁₅₀ SEQ ID NO: 43 30 Pprotein-10copy-Aβ1-12-loop2N₁₅₀ SEQ ID NO: 44 31 Pprotein-20copy-Aβ1-12-loop2N₁₅₀ SEQ ID NO: 45 32 Pprotein-40copy-Aβ1-12-loop2N₁₅₀ SEQ ID NO: 46 33 Pprotein-1copy-Aβ1-15-loop2D₁₅₁ SEQ ID NO: 47 34 Pprotein-10copy-Aβ1-15-loop2D₁₅₁ SEQ ID NO: 48 35 Pprotein-10copy-Aβ1-15-loop2D₁₅₁ SEQ ID NO: 49 36 Pprotein-40copy-Aβ1-15-loop2D₁₅₁ SEQ ID NO: 50 37 Pprotein-1copy-Aβ1-12-loop3D₁₆₈ SEQ ID NO: 5l 38 Pprotein-10copy-Aβ1-12-loop3D₁₆₈ SEQ ID NO: 52 39 Pprotein-20copy-Aβ1-12-loop3D₁₆₈ SEQ ID NO: 53 40 Pprotein-40copy-Aβ1-12-loop3D₁₆₈ SEQ ID NO: 54 41 Pprotein-1copy-Aβ1-6-loop3G₁₆₉ SEQ ID NO: 55 42 Pprotein-10copy-Aβ1-6-loop3G₁₆₉ SEQ ID NO: 56 43 Pprotein-20copy-Aβ1-6-loop3G₁₆₉ SEQ ID NO: 57 44 Pprotein-40copy-Aβ1-6-loop3G₁₆₉ SEQ ID NO: 58 45 Pprotein-1copy-Aβ1-15-loop3S₁₇₀ SEQ ID NO: 59 46 Pprotein-10copy-Aβ1-15-loop3S₁₇₀ SEQ ID NO: 60 47 Pprotein-20copy-Aβ1-15-loop3S₁₇₀ SEQ ID NO: 61 48 Pprotein-40copy-Aβ1-15-loop3S₁₇₀ SEQ ID NO: 62 49 Pprotein-1copy-Aβ1-9-loop3T₁₇₁ SEQ ID NO: 63 50 Pprotein-10copy-Aβ1-9-loop3T₁₇₁ SEQ ID NO: 64 51 Pprotein-20copy-Aβ1-9-loop3T₁₇₁ SEQ ID NO: 65 52 Pprotein-40copy-Aβ1-9-loop3T₁₇₁ SEQ ID NO: 66 53 Pprotein-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉ SEQ ID NO: 67 54 Pprotein-10copy-Aβ1-9-loop1G₇₂-10copy-Aβ1-9-loop2S₁₄₉ SEQ ID NO: 68 55 Pprotein-20copy-Aβ1-12-loop1G₇₂-20copy-Aβ1-12-loop2S₁₄₉ SEQ ID NO: 69 56P protein-40copy-Aβ1-15-loop1G₇₂-40copy-Aβ1-15-loop2S₁₄₉ SEQ ID NO: 7057 P protein-1copy-Aβ1-6-loop1I₇₀-1copy-Aβ1-12-loop2T₁₄₈ SEQ ID NO: 7158 P protein-3copy-Aβ1-9-loop1A₇₁-3copy-Aβ1-6-loop2S₁₄₉ SEQ ID NO: 72 59P protein-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉ SEQ ID NO: 73 60P protein-20copy-Aβ1-9-loop1G₇₂-20copy-Aβ1-15-loop2N₁₅₀ SEQ ID NO: 74 61P protein-30copy-Aβ1-15-loop1T₇₃-30copy-Aβ1-12-loop2D₁₅₁ SEQ ID NO: 7562 P protein-40copy-Aβ1-12-loop1Q₇₄-40copy-Aβ1-9-loop2S₁₄₉ SEQ ID NO: 7663 P protein-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉ SEQ ID NO: 7764 P protein-10copy-Aβ1-9-loop1G₇₂-20copy-Aβ1-9-loop2S₁₄₉ SEQ ID NO: 7865 P protein-20copy-Aβ1-12-loop1G₇₂-40copy-Aβ1-12-loop2S₁₄₉ SEQ ID NO:79 66 P protein-40copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-15-loop2S₁₄₉ SEQ IDNO: 80 67 P protein-1copy-Aβ1-6-loop1I₇₀-10copy-Aβ1-12-loop2T₁₄₈ SEQ IDNO: 81 68 P protein-10copy-Aβ1-9-loop1A₇₁-20copy-Aβ1-6-loop2S₁₄₉ SEQ IDNO: 82 69 P protein-20copy-Aβ1-15-loop1G₇₂-40copy-Aβ1-6-loop2S₁₄₉ SEQ IDNO: 83 70 P protein-40copy-Aβ1-9-loop1G₇₂-20copy-Aβ1-15-loop2N₁₅₀ SEQ IDNO: 84 71 P protein-20copy-Aβ1-15-loop1T₇₃-10copy-Aβ1-12-loop2D₁₅₁ SEQID NO: 85 72 P protein-10copy-Aβ1-12-loop1Q₇₄-1copy-Aβ1-9-loop2S₁₄₉ SEQID NO: 86 73 P protein-1copy-Aβ1-15-loop2S₁₄₉-1copy-Aβ1-15-loop3G₁₆₉ SEQID NO: 87 74 P protein-10copy-Aβ1-12-loop2S₁₄₉-10copy-Aβ1-12-loop3G₁₆₉SEQ ID NO: 88 75 P protein-20copy-Aβ1-6-loop2S₁₄₉-20copy-Aβ1-6-loop3G₁₆₉SEQ ID NO: 89 76 P protein-40copy-Aβ1-9-loop2S₁₄₉-40copy-Aβ1-9-loop3G₁₆₉SEQ ID NO: 90 77 P protein-1copy-Aβ1-6-loop2T₁₄₈-1copy-Aβ1-9-loop3D₁₆₈SEQ ID NO: 91 78 P protein-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉SEQ ID NO: 92 79 Pprotein-10copy-Aβ1-9-loop2S₁₄₉-10copy-Aβ1-15-loop3G₁₆₉ SEQ ID NO: 93 80P protein-20copy-Aβ1-15-loop2S₁₄₉-20copy-Aβ1-6-loop3G₁₆₉ SEQ ID NO: 9481 P protein-30copy-Aβ1-9-loop2N₁₅₀-30copy-Aβ1-12-loop3S₁₇₀ SEQ ID NO:95 82 P protein-40copy-Aβ1-15-loop2D₁₅₁-40copy-Aβ1-12-loop3T₁₇₁ SEQ IDNO: 96 83 P protein-1copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉ SEQ IDNO: 97 84 P protein-10copy-Aβ1-9-loop2S₁₄₉-20copy-Aβ1-9-loop3G₁₆₉ SEQ IDNO: 98 85 P protein-20copy-Aβ1-12-loop2S₁₄₉-40copy-Aβ1-12-loop3G₁₆₉ SEQID NO: 99 86 P protein-40copy-Aβ1-15-loop2S₁₄₉-10copy-Aβ1-15-loop2G₁₆₉SEQ ID NO: 100 87 P protein-1copy-Aβ1-6-loop2T₁₄₈-10copy-Aβ1-9-loop3D₁₆₈SEQ ID NO: 101 88 Pprotein-10copy-Aβ1-15-loop2N₁₅₀-20copy-Aβ1-6-loop3S₁₇₀ SEQ ID NO: 102 89P protein-20copy-Aβ1-l2-loop2S₁₄₉-40copy-Aβ1-15-loop3G₁₆₉ SEQ ID NO: 10390 P protein-40copy-Aβ1-15-loop2D₁₅₁-20copy-Aβ1-12-loop3T₁₇₁ SEQ ID NO:104 91 P protein-20copy-Aβ1-12-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉ SEQ IDNO: 105 92 P protein-10copy-Aβ1-9-loop2S₁₄₉-1copy-Aβ1-15-loop3G₁₆₉ SEQID NO: 106 93 P protein-1copy-Aβ1-15-loop1G₇₂-1copy-Aβ1-15-loop3G₁₆₉ SEQID NO: 107 94 P protein-10copy-Aβ1-9-loop1G₇₂-10copy-Aβ1-9-loop3G₁₆₉ SEQID NO: 108 95 P protein-20copy-Aβ1-6-loop1G₇₂-20copy-Aβ1-6-loop3G₁₆₉ SEQID NO: 109 96 P protein-40copy-Aβ1-12-loop1G₇₂-40copy-Aβ1-12-loop3G₁₆₉SEQ ID NO: 110 97 P protein-1copy-Aβ1-6-loop1₁₇₀-1copy-Aβ1-9-loop3D₁₆₈SEQ ID NO: 111 98 P protein-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-12-loop3G₁₆₉SEQ ID NO: 112 99 Pprotein-10copy-Aβ1-9-loop1A₇₁-10copy-Aβ1-15-loop3G₁₆₉ SEQ ID NO: 113 100P protein-20copy-Aβ1-12-loop1G₇₂-20copy-Aβ1-9-loop3S₁₇₀ SEQ ID NO: 114101 P protein-30copy-Aβ1-12-loop1Q₇₄-30copy-Aβ1-15-loop3T₁₇₁ SEQ ID NO:115 102 P protein-40copy-Aβ1-15-loop1T₇₃-40copy-Aβ1-6-loop3G₁₆₉ SEQ IDNO: 116 103 P protein-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉ SEQ IDNO: 117 104 P protein-10copy-Aβ1-9-loop1G₇₂-20copy-Aβ1-9-loop3G₁₆₉ SEQID NO: 118 105 P protein-20copy-Aβ1-12-loop1G₇₂-40copy-Aβ1-12-loop3G₁₆₉SEQ ID NO: 119 106 Pprotein-40copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-15-loop3G₁₆₉ SEQ ID NO: 120107 P protein-1copy-Aβ1-6-loop1I₇₀-10copy-Aβ1-9-loop3D₁₆₈ SEQ ID NO: 121108 P protein-3copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-12-loop3G₁₆₉ SEQ ID NO:122 109 P protein-10copy-Aβ1-12-loop1G₇₂-1copy-Aβ1-15-loop3S₁₇₀ SEQ IDNO: 123 110 P protein-20copy-Aβ1-9-loop1A₇₁-40copy-Aβ1-15-loop3G₁₆₉ SEQID NO: 124 111 P protein-40copy-Aβ1-9-loop1Q₇₄-20copy-Aβ1-12-loop3T₁₇₁SEQ ID NO: 125 112 Pprotein-30copy-Aβ1-15-loop1T₇₃-15copy-Aβ1-6-loop3G₁₆₉ SEQ ID NO: 126 113P protein-1copy-Aβ1-6-loop1I₇₀-10copy-Aβ1-9-loop2T₁₄₈-20copy- SEQ ID NO:127 Aβ1-12-looρ3D₁₆₈ 114 Pprotein-10copy-Aβ1-15-loop1A₇₁-20copy-Aβ1-12-loop2S₁₄₉- SEQ ID NO: 12840copy-Aβ1-9-loop3G₁₆₉ 115 Pprotein-1copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-9-loop2N₁₅₀- SEQ ID NO: 12910copy-Aβ1-15-loop3S₁₇₀ 116 Pprotein-10copy-Aβ1-6-loop1Q₇₄-30copy-Aβ1-12-loop2D₁₅₁- SEQ ID NO: 13020copy-Aβ1-15-loop3T₁₇₃ 117 Pprotein-10copy-Aβ1-6-loop1T₇₃-10copy-Aβ1-6-loop2S₁₄₉- SEQ ID NO: 13110copy-Aβ1-6-loop3G₁₆₉ 118 Pprotein-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉- SEQ ID NO: 1321copy-Aβ1-6-loop3G₁₆₉ 119 Pprotein-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉- SEQ ID NO: 1333copy-Aβ1-6-loop3G₁₆₉ 120 Pprotein-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉- SEQ ID NO: 13410copy-Aβ1-6-loop3G₁₆₉ 121 Pprotein-40copy-Aβ1-6-loop1G₇₂-40copy-Aβ1-6-loop2S₁₄₉- SEQ ID NO: 13540copy-Aβ1-6-loop3G₁₆₉ 122 P protein-10copy-Aβ1-6-loop2S₁₄₉(GGA)₀ SEQ IDNO: 136 123 P protein-10copy-Aβ1-6-loop2S₁₄₉(GGA)₁ SEQ ID NO: 137 124 Pprotein-10copy-Aβ1-6-loop2S₁₄₉(GGA)₅ SEQ ID NO: 138 125 Pprotein-10copy-Aβ1-6-loop2S₁₄₉(GGA)₁₀ SEQ ID NO: 139 126 Pprotein-10copy-Aβ1-6-loop2S₁₄₉(GGC)₃ SEQ ID NO: 140 127 Pprotein-1copy-Aβ1-6-loop2T₁₄₈-1copy-Aβ1-6-loop2S₁₄₉-1copy- SEQ ID NO:141 Aβ1-6-loop2N₁₅₀

The number of the recombinant P protein or recombinant P particle in thefollowing tables respectively corresponds to the corresponding number ofrecombinant P protein in Table 1.

The present invention also provides a method for preparing saidrecombinant P protein particles, comprising the following steps:

1. Synthesizing artificially a DNA fragment comprising a DNA fragmentencoding loop1, loop2 and/or loop3 domain(s) of a P protein embeddedwith multiple copies of Aβ1-m peptide;

2. Constructing pET26b-P protein plasmid (as shown in FIG. 1A);

3. Carrying out site-directed mutation at the position before loop1 andbehind loop3 of P protein by a point mutation method on condition thatthe amino acid sequence remains unchanged, to obtain the new restrictionenzyme sites mKpnI and mEagI (as shown in FIG. 1B);

4. According to various construction requirements, replacing multiplewild-type circular DNA in its entirety with the synthetic DNA fragmentencoding loop1, loop2 and/or loop3 domain(s) of a P protein embeddedwith multiple copies of Aβ1-m peptide obtained in step 1 by using therestriction enzyme site SalI in the nucleic acid encoding P protein andthe obtained enzyme sites mKpnI and mEagI in order to construct variousrecombinant P protein expression plasmids carrying multiple copies ofAβ1-m peptide respectively (as shown in FIG. 1D-1M);

5. Transferring the expression plasmids obtained in step 4 intoEscherichia coli, where the P protein embedded with an Aβ1-m immunogenis stably expressed and self-assembles into a P particle in the form ofa tetracosamer.

Furthermore, according to the present invention, the following analysisand verification experiments were carried out:

1. Ten preferred protein vaccines were characterized by particlediameter determination and electron microscope analysis (as shown inFIG. 4).

2. Experiments were carried out with different immune dosages anddifferent immune adjuvants in a C57BL/6J mouse model, usingPP-10copy-Aβ1-6-loop2S₁₄₉ protein vaccine. Results show that when CpG isused as the immune adjuvant, 25 μg protein vaccine could stimulate themouse to produce the highest titer of a specific antibody againstAβ1-42, and meanwhile would not induce the mouse to produce T cellresponses against Aβ1-42 (as shown in FIG. 5).

3. Using PP-1copy-Aβ1-6-loop2S₁₄₉, PP-10copy-Aβ1-6-loop2S₁₄₉ andPP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ asthe immunogens, and CpG as the immune adjuvant, the immunologicaleffects of the three protein vaccines were compared in mouse models, andthe effects of both subcutaneous and nasal immunization on theimmunological effects of the protein vaccines were analyzed (as shown inFIG. 6). Experimental results show that compared with nasalimmunization, subcutaneous immunization is more beneficial for theprotein vaccines to induce antibodies.

4. Immunization was carried out with 127 candidate proteins. Variousproteins were compared for their immunological effects, and the optimalcandidate vaccine was screened. Results show that various vaccines canstimulate the mouse to produce a specific antibody against Aβ1-42compared with the PBS control group; wherein the ten proteins,PP-10copy-Aβ1-6-loopG₇₂, PP-1copy-Aβ1-6-loop2S₁₄₉,PP-10copy-Aβ1-6-loop2S₁₄₉, PP-20copy-Aβ1-6-loop3G₁₆₉,PP-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉,PP-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉,PP-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉,PP-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉,PP-3 copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉,PP-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉,have the optimal immunological effects and can stimulate the productionof a high concentration of Aβ1-42.

Example 1. Construction of pET26b-P Protein Plasmid

1 μL of Nde I enzyme and 1 μL of Xho I enzyme (purchased from TakaraCorporation), and 5 μL of enzyme cleavage buffer (purchased from TakaraCorporation) were respectively added to 4 μg of pET26b vector plasmid(purchased from Novagen Corporation), and finally sterile water wasadded to the system to reach a final volume of 50 μL. Digestion wascarried out at 37° C. for 5 hours. Then the products were subjected toagarose gel electrophoresis, and recovered using a recovery column(purchased from Invitrogen) to obtain a plasmid vector having doubleenzyme-digested sticky ends.

A P protein nucleotide sequence as shown in SEQ ID NO: 9 was synthesizedby gene synthetic method. The synthetic gene fragment was subjected toNde I/Xho I double-enzyme digestion using the same method as mentionedabove. The products were subjected to agarose gel electrophoresis, andrecovered using a recovery column to obtain a gene fragment havingdouble enzyme-digested sticky ends.

The above double enzyme-digested vector fragment and fragment ofinterest (with a molar ratio of 1:3, and a total volume of 15 μL) weremixed, and 0.75 μL of T4 ligase (purchased from Takara Corporation) and1.5 μL of ligase buffer (purchased from Takara Corporation) were added.The ligation was carried out at 16° C. overnight to obtain a pET26b-Pprotein plasmid that can express a P protein particle, as shown in FIG.1A.

Example 2. Construction of pET26b-P Protein Plasmid with RestrictionEnzyme Sites mKpnI and mEagI

The site-directed mutation method was as follows:

1. 5′CCGCCG3′ was mutated to 5′CGGCCG3′ by site-directed mutation methodusing the pET26b-P protein plasmid constructed in Example 1 to obtainpET26b-P protein-mEagI plasmid containing a mEagI restriction enzymesite without altering the amino acid sequence. The specific method wasas follows: a pair of perfectly complementary bidirectional primerscontaining the mutation site was used:

(forward): SEQ ID NO: 11 5′CGTTCACTTGGCTCCGGCCGTGGCTCCAACC3′, and(reverse): SEQ ID NO: 12 5′GGTTGGAGCCACGGCCGGAGCCAAGTGAACG3′;the entire plasmid was subjected to PCR reaction, wherein the PCRreaction system was a KOD-Plus DNA polymerase system (purchased fromTOYOBO Corporation), and the total volume of the reaction system was 50μL (5 μL buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 μM upstreamprimer and 0.3 NM downstream primer, 50 ng template DNA, 1 μL KODenzyme, and water which was added to a final volume of 50 μL). PCR wascarried out in accordance with the reaction system instructions toobtain 20 μL of PCR products. 1 μL of DpnI enzyme (purchased from NEBCorporation) was added to the PCR products. Digestion was carried out at37° C. for 1 h. 10 μL of the digested products were added to Trant-Bluecompetent cells (purchased from Peking TransGen Biotech, Ltd.), andplaced on ice for 30 min. After that, the cells were subjected to heatshock at 42° C. for 45 s, placed on ice for 2 min. and then were addedto 600 μL of fluid LB medium without resistance, and recovered at 200rpm at 37° C. for 1 h. The culture broth was plated on a LB solidculture plate containing kanamycin (15 μg/mL), and were placed upsidedown at 37° C. overnight. The obtained mutant plasmid clones wereverified by sequencing. pET26b-P protein-mEagI plasmid is shown as FIG.1B.

2. 5′GGCACA3′ was mutated to 5′GGTACC3′ using the pET26b-P protein-mEagIrecombinant plasmid (as shown in FIG. 1B) constructed in the previousstep to obtain pET26b-P protein-mEagI&mKpnI plasmid containing both amEagI restriction enzyme site and a mKpnI restriction enzyme sitewithout altering the amino acid sequence. The specific method was asfollows:

a pair of perfectly complementary bidirectional primers containing themutation site was used:

(forward): SEQ ID NO: 13 5′GGTGTCCTGCTCGGTACCACCCAGCTCTCACC3′, and(reverse): SEQ ID NO: 14 5′GGTGAGAGCTGGGTGGTACCGAGCAGGACACC3′;and pET26b-P protein-mEagI&mKpnI plasmid was obtained by the sameconstruction method as mentioned above and verified by sequencing.pET26b-P protein-mEagI&mKpnI plasmid is shown as in FIG. 1C.

Example 3. Synthesis of P Particle Loop Gene Fragments of InterestEmbedded with Human Aβ1-m Genes

This step involves three different synthesis schemes in total:

The first is a P particle loop gene fragment of interest embedded with ahuman Aβ1-m gene located between a mKpnI restriction enzyme site and aSalI restriction enzyme site. That is, N1 copies of Aβ1-m gene aremerely embedded into loop1. The P protein prepared by this method isabbreviated as P protein-N 1copy-Aβ1-m-loop1.

The second is a P particle loop gene fragment of interest embedded witha human Aβ1-m gene located between a SalI restriction enzyme site and amEagI restriction enzyme site. That is, (1) N2 copies of Aβ1-m gene aremerely embedded into loop2, and the P protein prepared by this method isabbreviated as P protein-N2copy-Aβ1-m-loop2; (2) N3 copies of Aβ1-m geneare merely embedded into loop3, and the P protein prepared by thismethod is abbreviated as P protein-N3copy-Aβ1-m-loop3; and (3) N2 copiesof Aβ1-m gene are embedded into loop2 and N3 copies of Aβ1-m gene areembedded into loop3, and the P protein prepared by this method isabbreviated as P protein-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3, whereineach m is independently selected from an integer ranging from 1 to 40.

The third is a P particle loop gene fragment of interest embedded with ahuman Aβ1-m gene located between a mKpnI restriction enzyme site and amEagI restriction enzyme site. That is, (1) N1 copies of Aβ1-m gene areembedded into loop1 and N2 copies of Aβ1-m gene are embedded into loop2,and the P protein prepared by this method is abbreviated asprotein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2; (2) N1 copies of Aβ1-mgene are embedded into loop1 and N3 copies of Aβ1-m gene are embeddedinto loop3, and the P protein prepared by this method is abbreviated asP protein-N1copy-Aβ1-m-loop1-N3copy-Aβ1-m-loop3; and (3) N1 copies ofAβ1-m gene are embedded into loop1, N2 copies of Aβ1-m gene are embeddedinto loop2 and N3 copies of Aβ1-m gene are embedded into loop3, and theP protein prepared by this method is abbreviated as Pprotein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2-N3 copy-Aβ1-m-loop3,wherein each m is independently selected from an integer ranging from 1to 40.

The above three synthesis schemes are illustrated as follows:

3.1 Scheme I:

3.1.1

The loop1 gene fragment embedded with DNA encoding 10 copies of Aβ1-6peptide of P protein-10copy-Aβ1-6-loop1 G₇₂ (as shown in FIG. 2B) wassynthesized. The sequence of the synthetic gene fragment is shown as SEQID NO: 143.

3.2 Scheme II

3.2.1

The loop2 gene fragment embedded with DNA encoding one copy of Aβ1-6peptide of P protein-1copy-Aβ1-6-loop2S₁₄₉ (as shown in FIG. 2C) wassynthesized. The sequence of the synthetic gene fragment is shown as SEQID NO: 144.

3.2.2

The loop2 gene fragment embedded with DNA encoding 10 copies of Aβ1-6peptide of P protein-10copy-Aβ1-6-loop2S₁₄₉ (as shown in FIG. 2D) wassynthesized. The sequence of the synthetic gene fragment is shown as SEQID NO: 145.

3.2.3

The loop3 gene fragment embedded with DNA encoding 20 copies of Aβ1-6peptide of P protein-20copy-Aβ1-6-loop3G₁₆₉ (as shown in FIG. 2E) wassynthesized. The sequence of the synthetic gene fragment is shown as SEQID NO: 146.

3.2.4

The loop2 gene fragment embedded with DNA encoding 3 copies of Aβ1-12peptide and loop3 gene fragment embedded with DNA encoding 3 copies ofAβ1-6 peptide of P protein-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉(as shown in FIG. 2G) were synthesized. The sequence of the syntheticgene fragments is shown as SEQ ID NO: 147.

3.3 Scheme III

3.3.1

The loop1 gene fragment embedded with DNA encoding 10 copies of Aβ1-15peptide and loop2 gene fragment embedded with DNA encoding 10 copies ofAβ1-6 peptide of P protein-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉(as shown in FIG. 2F) were synthesized. The sequence of the syntheticgene fragments is shown as SEQ ID NO: 148.

3.3.2

The loop1 gene fragment embedded with DNA encoding 1 copy of Aβ1-6peptide and loop3 gene fragment embedded with DNA encoding 10 copies ofAβ1-6 peptide of P protein-1copy-A61-6-loop1 G₇₂-10copy-Aβ1-6-loop3S₁₆₉(as shown in FIG. 2H) were synthesized. The sequence of the syntheticgene fragments is shown as SEQ ID NO: 149.

3.3.3

The loop123 gene fragment embedded respectively with DNA encoding 1 copyof Aβ1-6 peptide of Pprotein-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉(as shown in FIG. 2I) was synthesized. The sequence of the syntheticgene fragment is shown as SEQ ID NO: 150.

3.3.4

The loop1, loop2, loop3 gene fragments embedded respectively with DNAencoding 3 copies of Aβ1-6 peptide of Pprotein-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉(as shown in FIG. 2J) were synthesized. The sequence of the syntheticgene fragments is shown as SEQ ID NO: 151.

3.3.5

The loop1, loop2, loop3 gene fragments embedded respectively with DNAencoding 10 copies of Aβ1-6 peptide ofPprotein-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉(as shown in FIG. 2K) were synthesized. The sequence of the syntheticgene fragments is shown as SEQ ID NO: 152.

Furthermore, in addition to the above 10 recombinant P proteins,synthetic methods of other 117 recombinant P proteins respectivelyinvolved the above three schemes, as specifically shown in Table 2.

TABLE 2 Synthetic methods of 127 recombinant P proteins and constructionmethods of 127 plasmids expressing the recombinant P proteins accordingto the invention Construction methods Synthetic methods of of plasmidsexpressing recombinant P proteins recombinant P proteins Similar SimilarNO. Scheme Scheme Scheme Scheme 1 1 3.1.1 1 4.1.1 2 1 3.1.1 1 4.1.1 3 13.1.1 1 4.1.1 4 1 3.1.1 1 4.1.1 5 1 3.1.1 1 4.1.1 6 1 3.1.1 1 4.1.1 7 13.1.1 1 4.1.1 8 1 3.1.1 1 4.1.1 9 1 3.1.1 1 4.1.1 10 1 3.1.1 1 4.1.1 111 3.1.1 1 4.1.1 12 1 3.1.1 1 4.1.1 13 1 3.1.1 1 4.1.1 14 1 3.1.1 1 4.1.115 1 3.1.1 1 4.1.1 16 1 3.1.1 1 4.1.1 17 1 3.1.1 1 4.1.1 28 1 3.1.1 14.1.1 19 1 3.1.1 1 4.1.1 20 1 3.1.1 1 4.1.1 21 2 3.2.1 2 4.2.1 22 23.2.1 2 4.2.1 23 2 3.2.1 2 4.2.1 24 2 3.2.1 2 4.2.1 25 2 3.2.1 2 4.2.126 2 3.2.1 2 4.2.1 27 2 3.2.1 2 4.2.1 28 2 3.2.1 2 4.2.1 29 2 3.2.1 24.2.1 30 2 3.2.1 2 4.2.1 31 2 3.2.1 2 4.2.1 32 2 3.2.1 2 4.2.1 33 23.2.1 2 4.2.1 34 2 3.2.1 2 4.2.1 35 2 3.2.1 2 4.2.1 36 2 3.2.1 2 4.2.137 2 3.2.3 2 4.2.3 38 2 3.2.3 2 4.2.3 39 2 3.2.3 2 4.2.3 40 2 3.2.3 24.2.3 41 2 3.2.3 2 4.2.3 42 2 3.2.3 2 4.2.3 43 2 3.2.3 2 4.2.3 44 23.2.3 2 4.2.3 45 2 3.2.3 2 4.2.3 46 2 3.2.3 2 4.2.3 47 2 3.2.3 2 4.2.348 2 3.2.3 2 4.2.3 49 2 3.2.3 2 4.2.3 50 2 3.2.3 2 4.2.3 51 2 3.2.3 24.2.3 52 2 3.2.3 2 4.2.3 53 3 3.3.1 3 4.3.1 54 3 3.3.1 3 4.3.1 55 33.3.1 3 4.3.1 56 3 3.3.1 3 4.3.1 57 3 3.3.1 3 4.3.1 58 3 3.3.1 3 4.3.159 3 3.3.1 3 4.3.1 60 3 3.3.1 3 4.3.1 61 3 3.3.1 3 4.3.1 62 3 3.3.1 34.3.1 63 3 3.3.1 3 4.3.1 64 3 3.3.1 3 4.3.1 65 3 3.3.1 3 4.3.1 66 33.3.1 3 4.3.1 67 3 3.3.1 3 4.3.1 68 3 3.3.1 3 4.3.1 69 3 3.3.1 3 4.3.170 3 3.3.1 3 4.3.1 71 3 3.3.1 3 4.3.1 72 3 3.3.1 3 4.3.1 73 2 3.2.4 24.3.1 74 2 3.2.4 2 4.3.1 75 2 3.2.4 2 4.3.1 76 2 3.2.4 2 4.3.1 77 23.2.4 2 4.3.1 78 2 3.2.4 2 4.3.1 79 2 3.2.4 2 4.3.1 80 2 3.2.4 2 4.3.181 2 3.2.4 2 4.3.1 82 2 3.2.4 2 4.3.1 83 2 3.2.4 2 4.3.1 84 2 3.2.4 24.3.1 85 2 3.2.4 2 4.3.1 86 2 3.2.4 2 4.3.1 87 2 3.2.4 2 4.3.1 88 23.2.4 2 4.3.1 89 2 3.2.4 2 4.3.1 90 2 3.2.4 2 4.3.1 91 2 3.2.4 2 4.3.192 2 3.2.4 2 4.3.1 93 3 3.3.2 3 4.3.1 94 3 3.3.2 3 4.3.1 95 3 3.3.2 34.3.1 96 3 3.3.2 3 4.3.1 97 3 3.3.2 3 4.3.1 98 3 3.3.2 3 4.3.1 99 33.3.2 3 4.3.1 100 3 3.3.2 3 4.3.1 101 3 3.3.2 3 4.3.1 102 3 3.3.2 34.3.1 103 3 3.3.2 3 4.3.1 104 3 3.3.2 3 4.3.1 105 3 3.3.2 3 4.3.1 106 33.3.2 3 4.3.1 107 3 3.3.2 3 4.3.1 108 3 3.3.2 3 4.3.1 109 3 3.3.2 34.3.1 110 3 3.3.2 3 4.3.1 111 3 3.3.2 3 4.3.1 112 3 3.3.2 3 4.3.1 113 33.3.3 3 4.3.1 114 3 3.3.3 3 4.3.1 115 3 3.3.3 3 4.3.1 116 3 3.3.3 34.3.1 117 3 3.3.3 3 4.3.1 118 3 3.3.3 3 4.3.1 119 3 3.3.3 3 4.3.1 120 33.3.3 3 4.3.1 121 3 3.3.3 3 4.3.1 122 2 3.2.1 2 4.2.1 123 2 3.2.1 24.2.1 124 2 3.2.1 2 4.2.1 125 2 3.2.1 2 4.2.1 126 2 3.2.1 2 4.2.1 127 23.2.1 2 4.2.1

Example 4. Construction of a pET26b Vector Stably Expressing a P ProteinEmbedded with an Aβ1-m Immunogen

Corresponding to Example 3, this step involves three different synthesisschemes in total:

The first, directed at Scheme I of Example 3, is a P particle loop genefragment of interest embedded with a human Aβ1-m gene located between amKpnI restriction enzyme site and a SalI restriction enzyme site. Thatis, N1 copies of API-m gene are merely embedded into loop1. The Pprotein prepared by this method is abbreviated as Pprotein-N1copy-Aβ1-m-loop1.

The second, directed at Scheme II of Example 3, is a P particle loopgene fragment of interest embedded with a human API-m gene locatedbetween a SalI restriction enzyme site and a mEagI restriction enzymesite. That is, (1) N2 copies of Aβ1-m gene are merely embedded intoloop2, and the P protein prepared by this method is abbreviated as Pprotein-N2copy-Aβ1-m-loop2; (2) N3 copies of Aβ1-m gene are merelyembedded into loop3, and the P protein prepared by this method isabbreviated as P protein-N3copy-Aβ1-m-loop3; and (3) N2 copies of Aβ1-mgene are embedded into loop2 and N3 copies of Aβ1-m gene are embeddedinto loop3, and the P protein prepared by this method is abbreviated asP protein-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3.

The third, directed at Scheme III of synthesizing a P protein embeddedwith an API-m immunogen in Example 3, is a P particle loop gene fragmentof interest embedded with a human API-m gene located between a mKpnIrestriction enzyme site and a mEagI restriction enzyme site. That is,(1) N1 copies of Aβ1-m gene are embedded into loop1 and N2 copies ofAβ1-m gene are embedded into loop2, and the P protein prepared by thismethod is abbreviated as Pprotein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2; (2) N1 copies of Aβ1-mgene are embedded into loop1 and N3 copies of Aβ1-m gene are embeddedinto loop3, and the P protein prepared by this method is abbreviated asP protein-N1copy-Aβ1-m-loop1-N3copy-Aβ1-m-loop3; and (3) N1 copies ofAβ1-m gene are embedded into loop1, N2 copies of Aβ1-m gene are embeddedinto loop2 and N3 copies of Aβ1-m gene are embedded into loop3, and theP protein prepared by this method is abbreviated as Pprotein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3.

The above three synthesis schemes are illustrated as follows:

4.1 Scheme I:

The pET26b-P protein-mEagI-mKpnI plasmid vector obtained in Example 2was subjected to SalI/KpnI double-enzyme digestion, using the mKpnIrestriction enzyme site obtained by mutation and the SalI restrictionenzyme site carried by the sequence itself, in order to excise theoriginal loop1 region. The plasmid was recovered as the vector.

Meanwhile, the recombinant loop1 DNA fragment comprising multiple copiesof the human Aβ1-m sequence synthesized in Example 3 was subjected toSalI/KpnI double-enzyme digestion in order to obtain the DNA fragment ofinterest.

The digested DNA fragment of interest was ligated to the digested vectorin order to obtain a pET26b plasmid that can express a recombinant Pprotein embedded with an Aβ1-m immunogen. Finally, the plasmid wasverified by sequencing, and thus a correct plasmid was obtained.

4.1.1 Construction of a plasmid vector expressingPP-10copy-Aβ1-6-loop1G₇₂ Protein

1 μL of Sal I enzyme and 1 μL of Kpn I enzyme (purchased from TakaraCorporation) and 5 μL of digestion buffer (purchased from TakaraCorporation) were added respectively to 4 μg of pET26b-Pprotein-mEagI-mKpnI vector plasmid obtained in Example 2, and finallysterile water was added to the system to reach a final volume of 50 μL.Digestion was carried out at 37° C. for 5 hours. The digested productswere subjected to agarose gel electrophoresis, and recovered using gelrecovery kit (purchased from Tiangen Biotech Co., Ltd.) to obtain aplasmid vector having sticky ends.

Meanwhile, the DNA fragment 10copy-Aβ1-6-loop1G₇₂ synthesized in Example3 was subjected to SalI/KpnI double-enzyme digestion by the same methodto obtain the gene fragment of interest.

The digested vector was mixed with the digested gene fragment ofinterest. 0.75 μL of T4 ligase (purchased from Takara Corporation) and1.5 μL of ligase buffer (purchased from Takara Corporation) were addedto the mixture. The ligation was carried out at 16° C. overnight toobtain a plasmid vector expressing PP-10copy-Aβ1-6-loop1G₇₂ protein. Theplasmid was verified by sequencing, as shown in FIG. 1D.

4.2 Scheme II

The pET26b-P protein-mEagI-mKpnI plasmid vector obtained in Example 2was subjected to SalI/EagI double-enzyme digestion, using the mKpnIrestriction enzyme site obtained by mutation and the SalI restrictionenzyme site carried by the sequence itself, in order to excise theoriginal loop2 and loop3 regions. The plasmid was recovered as thevector.

Meanwhile, the recombinant loop2 and loop3 DNA fragments comprisingmultiple copies of the human Aβ1-m sequence synthesized in Example 3 wassubjected to SalI/EagI double-enzyme digestion to obtain the DNAfragment of interest.

The digested DNA fragment of interest was ligated to the digested vectorto obtain a pET26b plasmid that can express a recombinant P proteinembedded with an Aβ1-m immunogen. Finally, the plasmid was verified bysequencing, and thus a correct plasmid was obtained.

4.2.1 Construction of a Plasmid Vector ExpressingPP-1Copy-Aβ1-6-loop2S₁₄₉ Protein

1 μL of SalI enzyme and 1 μL of EagI enzyme (purchased from TakaraCorporation) and an appropriate amount of digestion buffer (purchasedfrom Takara Corporation) were respectively added to 4 μg of the pET26b-Pprotein-mEagI-mKpnI plasmid vector obtained in Example 2, and finallysterile water was added to reach a final volume of 50 μL. Digestion wascarried out at 37° C. for 5 hours. The digested products were subjectedto agarose gel electrophoresis, and recovered using gel recovery kit(purchased from Tiangen Biotech Co., Ltd.) to obtain a plasmid vectorhaving sticky ends.

Meanwhile, the DNA fragment 1copy-Aβ1-6-loop2S₁₄₉ synthesized in Example3 was subjected to SalI/KpnI double-enzyme digestion by the same methodto obtain the gene fragment of interest.

The digested vector was mixed with the digested gene fragment ofinterest. 0.75 μL of T4 ligase (purchased from Takara Corporation) and1.5 μL of ligase buffer (purchased from Takara Corporation) were addedto the mixture. The ligation was carried out at 16° C. overnight toobtain a plasmid vector expressing PP-1copy-Aβ1-6-loop2S₁₄₉ protein. Theplasmid was verified by sequencing, as shown in FIG. 1E.

4.2.2 Construction of a Plasmid Vector ExpressingPP-10Copy-Aβ1-6-loop2S₁₄₉ Protein

A plasmid vector expressing PP-10copy-Aβ1-6-loop2S₁₄₉ protein wasconstructed, using the same method as 4.2.1. The plasmid is shown inFIG. 1F.

4.2.3 Construction of a Plasmid Vector ExpressingPP-20Copy-Aβ1-6-loop3G₁₆₉ Protein

A plasmid vector expressing PP-20copy-Aβ1-6-loop3G₁₆₉ protein wasconstructed, using the same method as 4.2.1. The plasmid is shown inFIG. 1G.

4.2.4 Construction of a Plasmid Vector ExpressingPP-3Copy-Aβ1-12-loopS₁₄₉-3Copy-Aβ1-6-loop3G₁₆₉ Protein

A plasmid vector expressingPP-3copy-Aβ1-12-loopS₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ protein was constructed,using the same method as 4.2.1. The plasmid is shown in FIG. 1I.

4.3 Scheme III

The pET26b-P protein-mEagI-mKpnI plasmid vector obtained in Example 2was subjected to mKpnI/mEagI double-enzyme digestion, using the mEagIrestriction enzyme site and the mKpnI restriction enzyme site obtainedby mutation, in order to excise the original loop1, loop2 and loop3regions. The plasmid was recovered as the vector.

Meanwhile, the recombinant loop1, loop2 and loop3 DNA fragmentscomprising multiple copies of the human Aβ1-m sequence synthesized inExample 3 was subjected to mKpnI/mEagI double-enzyme digestion in orderto obtain the DNA fragment of interest.

The digested DNA fragment of interest was ligated to the digested vectorin order to obtain a pET26b plasmid that can express a recombinant Pprotein embedded with an Aβ1-m immunogen. Finally, the plasmid wasverified by sequencing, and thus a correct plasmid was obtained.

4.3.1 Construction of a Plasmid Vector Expressing PP-3Copy-Aβ1-6-Loop1-G₇₂-3Copy-Aβ1-6-loop2S₁₄₉-3Copy-Aβ1-6-loop3G₁₆₉ Protein

1 μL of KpnI enzyme and 1 μL of EagI enzyme (purchased from TakaraCorporation) and an appropriate amount of digestion buffer (purchasedfrom Takara Corporation) were added respectively to 4 μg of the pET26b-Pprotein-mEagI-mKpnI vector plasmid obtained in Example 2, and finallysterile water was added to reach a final volume of 50 μL. Digestion wascarried out at 37° C. for 5 hours. The digested products were subjectedto agarose gel electrophoresis, and recovered using gel recovery kit(purchased from Tiangen Biotech Co., Ltd.) to obtain a plasmid vectorhaving sticky ends.

Meanwhile, the DNA fragment3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉synthesized in Example 3 was subjected to EagI/KpnI double-enzymedigestion by the same method to obtain the gene fragment of interest.

The digested vector was mixed with the digested gene fragment ofinterest. 0.75 μL of T4 ligase (purchased from Takara Corporation) and1.5 μL of ligase buffer (purchased from Takara Corporation) were addedto the mixture. The ligation was carried out at 16° C. overnight toobtain a plasmid vector expressing PP-1copy-Aβ1-6-loop2S₁₄₉ protein. Theplasmid was verified by sequencing, as shown in FIG. 1L.

4.3.2 Construction of a plasmid vector expressingPP-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉ protein

A plasmid vector expressingPP-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉ protein wasconstructed, using the same method as 4.3.1. The plasmid is shown inFIG. 1H.

4.3.3 Construction of a Plasmid Vector ExpressingPP-1Copy-Aβ1-6-loop1G₇₂-10Copy-Aβ1-6-loop3G₁₆₉ Protein

A plasmid vector expressingPP-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉ protein was constructed,using the same method as 4.3.1. The plasmid is shown in FIG. 1J.

4.3.4 Construction of a Plasmid Vector ExpressingPP-1Copy-Aβ1-6-loop1G₇₂-1Copy-Aβ1-6-loop2S₁₄₉-1Copy-Aβ1-6-loop3G₁₆₉Protein

A plasmid vector expressingPP-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-11copy-Aβ1-6-loop3G₁₆₉protein was constructed, using the same method as 4.3.1. The plasmid isshown in FIG. 1K.

4.3.5 Construction of a Plasmid Vector ExpressingPP-10Copy-Aβ1-6-loop1G₇₂-10Copy-Aβ1-6-loop2S₁₄₉-10 Copy-Aβ1-6-loop3G₁₆₉Protein

A plasmid vector expressingPP-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉protein was constructed, using the same method as 4.3.1. The plasmid isshown in FIG. 1M.

Furthermore, in addition to the above 10 recombinant P proteins,construction methods of other 117 plasmids expressing recombinant Pproteins respectively involve the above three schemes, specifically asshown in Table 2. The obtained recombinant plasmids are not shown.

Example 5 Expression and Purification of a P Protein Embedded with aHuman Aβ1-m Immunogen

5.1 Expression of a Recombinant P Particle Protein

1 μL of the recombinant plasmids prepared in the above examples wererespectively added to 100 μL of E. coli BL21 competent cells (purchasedfrom TransGen Corporation), ice-bathed for 30 min, heat shocked for 90 sin a water-bath at 42° C., and then ice-bathed for 2 min. 600 μL of LBmedium was added to the mixture, and cultured at 180 rpm/min at 37° C.for 1 h. The mixture was coated evenly on a LB solid medium containingkanamycin (15 μg/mL) resistance and cultured at 37° C. for 24 h toobtain strains that can stably express recombinant proteins. A growingcolony was picked and inoculated into 20 mL of LB medium. The mixturewas cultured at 220 rpm at 37° C. When the OD value of the culturemixture reached 1.0, induction by Isopropyl β-D-Thiogalactoside (IPTG ata final concentration of 0.33 mmol/L) was carried out at 220 rpm at 16°C. overnight. After the induction, the culture broth was centrifuged at4000 rpm for 20 min. The supernatant was discarded, and the bacterialprecipitates were resuspended with PBS. Centrifugation was conductedagain at 4000 rpm for 20 min and the supernatant was discarded to obtainthe bacterial precipitates containing proteins of interest.

5.2 Extraction and Purification of Recombinant P Particle Proteins

The bacterial precipitates obtained in 5.1 were resuspended by adding 20mL of protein buffer (pH8.0, containing 50 mM Tris and 300 mM KCl). Thebacteria were lysed by sonication on ice for 30 min. The mixture wascentrifuged at 12,000 rpm at 4° C. for 30 min. Subsequently, thesupernatant was taken and allowed to pass through 0.45 μm filtermembrane to obtain crude extracts of proteins.

The structures of recombinant P particle proteins are shown as FIG.3A-J.

The crude extracts of proteins were purified using a cation exchangecolumn (purchased from GE Corporation). The specific scheme was asfollows: First, the exchange column was rinsed with ultrapure water in avolume of about 100 mL, followed by equilibration with PB solution (pH5.0) at a flow rate of 2 mL/min. Then 20 mL of the crude extracts ofproteins were added to the exchange column at a flow rate of 1 ml/min.After the sample was completely loaded onto the column, the exchangecolumn was rinsed with PB solution (pH 7.0) to remove proteinimpurities, and then eluted with PB solution containing 1 mol/L NaCl.The proteins at peak value were collected to obtain the proteins ofinterest.

The proteins were further purified by a hydrophobic chromatographycolumn (purchased from GE Corporation). The specific scheme was asfollows: First, the column was rinsed with ultrapure water, and thenwith PB solution (pH 7.0) at a flow rate of 2 mL/min. After the columnwas equilibrated well, the protein sample was loaded onto it. After thesample was completely loaded onto the column, the column was eluted bygradient with PB (pH 7.0) and 1 mol/L NaCl solution for 2 hours. Theconcentration of NaCl decreases from 1 mol/L to 0.1 mol/L. The proteinswere collected at peak value.

The sizes of 10 P particle protein monomers were identified by reductiveSDS-PAGE. The upper panels of FIGS. 4A-J respectively show that the sizeof PP-10copy-Aβ1-6-loop1G72 protein is 45 KD; the size ofPP-1copy-Aβ1-6-loop2S₁₄₉ protein is 37 KD; the size ofPP-10copy-Aβ1-6-loopS₁₄₉ protein is 45 KD; the size ofPP-20copy-Aβ1-6-loop3G₁₆₉ protein is 55 KD; the size ofPP-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉ protein is 66 KD; thesize of PP-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ protein is 38KD; the size of PP-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉ proteinis 47 KD; the size ofPP-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉protein is 3 KD; the size ofPP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉protein is KD; and the size ofPP-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉is 66 KD.

Then the tetracosamers of P protein particles were further isolated andpurified using a Superdex 200 molecular sieve (purchased from GECorporation). The procedures were as follows: The column was rinsed withultrapure water at a flow rate of 1 mL/min for one volume of the column,and then again with about 120 mL of PB buffer (pH 5). After that, 2 mLof protein extraction solution was added to the column and washed withPB buffer at a flow rate of 1 mL/min. The proteins at peak value werecollected to obtain the tetracosamers of P particle proteins. Threeproteins were tested for their polymer structures by nativepolyacrylamide gel electrophoresis, as shown in the middle panels ofFIGS. 4A-J. All ten protein bands are above 225 KDa, which indicatesthat recombinant proteins can self-assemble into tetracosamers of Pprotein particles, and they can remain their polymer form after beingpurified.

Example 6 Characterization of a P Protein Particle Embedded with a HumanAβ1-m Immunogen

Inventors further analyzed the particle diameter and morphology of 10protein polymers. The particle diameter was tested using a nanoparticlediameter analyzer (purchased from Malvern Corporation) in accordancewith the instructions of the manufacturer. The analysis results showedthat there were particles having an average diameter of about 20 nm inthe solution of the above 10 recombinant P particles. As shown in thelower panels of FIGS. 4A-J, PP-10copy-Aβ1-6-loop1G₇₂ protein particlehas a particle diameter of 27.88 nm; PP-1copy-Aβ1-6-loop2S₁₄₉ proteinparticle has a particle diameter of 17.44 nm; PP-10copy-Aβ1-6-loop2S₁₄₉protein particle has a particle diameter of 27.64 nm;PP-20copy-Aβ1-6-loop3G₁₆₉ protein particle has a particle diameter of32.55 nm; PP-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉ proteinparticle has a particle diameter of 35.88 nm;PP-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉ protein particle has aparticle diameter of 17.02 nm;PP-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉ protein particle has aparticle diameter of 28.92 nm;PP-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3G₁₆₉protein particle has a particle diameter of 18.14 nm;PP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉protein particle has a particle diameter of 25.56 nm; andPP-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉protein particle has a particle diameter of 36.09 nm. Meanwhile, theresults of electron microscope tests show that the recombinant Pproteins are in the form of approximately spherical particles, and inthe form of polymers. According to the ruler, 10 protein polymers mainlycomprise particles of about 20 nm. Thus, all the above 10 recombinant Pproteins can self-assemble in vitro to form tetracosamers of Pparticles.

The sizes and P particle diameters of the 127 P particle proteinsaccording to the invention are shown as Table 3.

TABLE 3 The sizes and P particle diameters of 127 different forms ofrecombinant P particle proteins Size of protein P particle diameter NO.(KD) (nm) 1 36.2 17.75 2 48.4 31.23 3 68.2 36.37 4 114.4 39.86 5 36.616.98 6 51.7 31.53 7 74.8 38.13 8 74.8 38.2 9 36.2 16.98 10 45.1 27.8811 55.0 32.07 12 74.8 37.89 13 36.9 17.89 14 55.0 31.99 15 55.0 31.68 1688.0 39.54 17 37.2 17.89 28 45.1 18.03 19 61.6 34.66 20 101.2 39.75 2136.5 16.77 22 48.4 31.15 23 61.6 34.15 24 88.0 39.65 25 36.2 17.44 2645.1 27.64 27 55.0 31.97 28 74.8 38.17 29 36.9 17.95 30 51.7 31.67 3168.2 36.34 32 101.2 39.67 33 37.2 18.01 34 55.0 32.01 35 74.8 38.22 36114.4 39.92 37 36.9 18.02 38 51.7 31.42 39 68.2 36.41 40 101.2 40.11 4136.2 17.51 42 45.1 27.61 43 55.0 32.55 44 74.8 38.21 45 37.2 18.13 4655.0 31.87 47 74.8 38.17 48 114.4 40.17 49 36.5 18.16 50 48.4 31.26 5161.6 36.18 52 88.0 39.18 53 41.5 25.06 54 61.9 34.09 55 101.5 40.06 56193.9 45.3 57 38.2 19.2 58 42.5 26.2 59 65.2 35.88 60 101.5 39.83 61144.4 43.7 62 154.3 44.01 63 46.4 30.31 64 75.1 31.88 65 134.5 42.67 66134.5 43.12 67 53.0 31.99 68 68.5 36.28 69 114.7 39.67 70 127.9 40.02 7191.6 39.77 72 53.4 31.89 73 49.4 18.24 74 68.5 36.44 75 75.1 38.17 76141.1 42.55 77 37.8 17.1 78 43.5 17.02 79 68.5 35.21 80 94.9 39.62 81124.6 41.41 82 180.7 45.23 83 46.4 28.31 84 75.1 38.21 85 134.5 42.71 86134.5 41.93 87 49.7 31.19 88 75.1 38.38 89 147.7 44.22 90 147.7 43.91 9178.4 39.4 92 50.7 31.87 93 39.4 18.31 94 61.9 34.67 95 75.1 38.09 96167.5 45.8 97 37.8 18.45 98 43.4 27.67 99 68.5 36.38 100 94.9 40.01 101144.4 43.96 102 154.3 44.38 103 46.4 28.92 104 75.1 38.22 105 134.542.51 106 134.5 42.79 107 49.7 31.33 108 55.0 32.09 109 54.0 32.91 110141.1 43.68 111 121.3 40.03 112 109.8 38.88 113 83.1 39.44 114 141.543.21 115 60.6 35.87 116 134.9 42.57 117 65.6 36.12 118 38.8 18.14 11944.8 25.56 120 65.6 36.09 121 154.7 44.11 122 41.5 20.45 123 42.7 24.24124 47.5 28.99 125 53.6 32.01 126 45.1 27.64 127 38.9 18.15

3. Immunological Effects of Recombinant P Particle Protein Vaccines

3.1 Determination of Immune Dosages and Adjuvants of P Particle ProteinVaccines

The PP-10copy-Aβ1-6-loop2S₁₄₉ protein vaccine was selected for use infemale C57BL/6 mice aged 6-8 weeks (purchased from Beijing HFKBioscience Co., LTD) to determine the immune dosage and immune adjuvant.The immune dosages were respectively 12.5 μg, 25 μg and 50 μg/animal,and were increased to 100 μL/animal with sterile PBS. Each groupcontained 6 mice. The immunization was performed by subcutaneousinjection. The immune adjuvants were an aluminium adjuvant (purchasedfrom Brenntag Biosector Corporation) and an CpG adjuvant that canspecifically stimulate the body to produce humoral immunity (purchasedfrom Takara Corporation). The nucleotide sequence of CpG adjuvant was asfollows: TGTCGTCGTCGTTTGTCGTTTGTCGTT (SEQ ID NO: 153). The immunizationwas carried out at day 1, day 15 and day 29 (three times in total).Blood was taken on the day before every immunization. The mice weresacrificed two weeks after the third immunization. The recombinant Pparticle protein vaccine was tested for immunological responses of theinduced humoral immunity and cellular immunity in mice. There were 7test groups and 3 control groups. The immune dosage, adjuvant andimmunizing antigen for each group are shown in Table 4.

TABLE 4 Immunization scheme of PP-10copy-Aβ1-6-loop2S₁₄₉ Group Immunedosage (100 μl) Adjuvant Immunizing antigen Negative — — — control group— aluminium — adjuvant (200 μg) — CpG — (10 μg) Test group 12.5 μg —PP-10copy-Aβ1-6-loop2S₁₄₉ 25 μg — PP-10copy-Aβ1-6-loop2S₁₄₉ 50 μg —PP-10copy-Aβ1-6-loop2S₁₄₉ 25 μg aluminium PP-10copy-Aβ1-6-loop2S₁₄₉adjuvant (200 μg) 25 μg CpG PP-10copy-Aβ1-6-loop2S₁₄₉ (10 μg) 25 μgaluminium PP-10copy-Aβ1-6-loop2S₁₄₉ adjuvant + CpG (10 μg) 100 μgFreund's adjuvant Aβ1-42 (100 μl)

3.1.1 Humoral Immunity-ELISA Detection Experiment

Tails of the mice were cut and blood was taken on the day before everyimmunization. Blood samples were placed at 37° C. for 2 h, then placedat 4° C. for 1 h, and centrifuged at 3,000 rpm to take the supernatantserum. The serum was frozen for use. Aβ1-42 (purchased from GL Biochem(Shanghai) Ltd.) was used as antigen and formulated into 1 mg/mLsolution with sterile PBS. The solution was diluted to 1 ng/L withantigen coating solution and used for coating a 96-well plate (100μL/well). The plate was coated at 4° C. overnight. After each well waswashed three times with 300 μL of PBST (pH 7.4, 0.01 mol/L PBS,containing 0.05% Tween-20), blocking solution (pH 7.4, 0.01 mol/L PBS,20% fetal bovine serum) was added. Blocking was carried out at 37° C.for 2 hours. Each well was washed three times with PBST. To the wellswere added different dilution gradients (1:200, 1:800, 1:3200, 1:12800,1:51200 and 1:204800) of antiserum (100 μL/well), and incubation wascarried out at 37° C. for 1 hour. Each well was washed three times withPBST. To the wells were added 0.3 μg/ml of HPR (horse radish peroxidase)goat-anti-mouse secondary antibody (purchased from BeijingDingguochangsheng Biotechnology Co. LTD) (100 μL/well), and incubationwas carried out at 37° C. for 1 hour. Each well was washed three timeswith PBST. To the wells were added the substrate tetramethylbenzidine(TMB) (purchased from Tiangen Biotech Co., Ltd.) (100 μL/well), andcolor was developed in the dark for 25 min. 50 μL of 2M sulfuric acidwas added to each well to terminate the reaction. Absorbance wasdetected at 450 nm using a microplate reader (purchased from Bio-redCorporation).

Experimental results are shown in FIG. 5A. When CpG is used as immuneadjuvant and the immune dosage is 25 μg, or the immune dosage is 50 μgand no immune adjuvant is used, PP-10copy-Aβ1-6-loop2-S₁₄₉ proteinvaccine at all the shown dilutions can stimulate the mouse to producerelatively high titers of specific antibodies against Aβ42.

3.1.2 Cellular Immunity-ELISPOT Detection

A 96-well plate was coated with the monoclonal antibody against cytokineinterferon γ (from elispot kit purchased from BD Corporation) in aconcentration of 5 μg/mL (50 μL/well), and covered at 4° C. overnight.After discarding the coating antibody and washing once with a completemedium containing 10% fetal bovine serum, 200 μL of this complete mediumwas added to each well. Blocking was carried out at 37° C. for 1 hour,and then the medium was discarded. Mice used in the experiment weresacrificed by neck-pulling. Spleen cells of the mice were taken out andformulated into a cell suspension in a concentration of 10⁷/mL. The cellsuspension was added to the coated 96-well plate (100 μL/well). 100 μLof 1 μg/mL specific antigen Aβ1-42 was added to each well. The plate wasthen cultured at 37° C. in an incubator containing 5% CO₂ for 24 h tostimulate and activate the cells. 24 h later, the plate was washed twotimes with sterile water, and six times with sterile PBST (pH7.4, 0.01mol/L PBS, containing 0.05% Tween-20) buffer to wash the cells away. 50μL of 2 μg/mL antibody against interferon γ (from elispot kit purchasedfrom BD Corporation) was added to each well and incubated at roomtemperature for 2 hours. The 96-well plate was washed, and horse radishperoxidase labeled biotin secondary antibody (from elispot kit purchasedfrom BD Corporation) was added (50 μL/well). The plate was cultured atroom temperature for 2 h, and washed four times with PBST, and two timeswith PBS. 50 μL of Elispot color developing solution (AEC substrate) wasadded to each well and reacted in the dark at room temperature for 5-60min. The staining solution was discarded, and the plate was washed withdistilled water. After being dried overnight, the sample was calculatedfor the number of activated cells using a microscope.

The results are shown in FIG. 5B. In the T cell response-positivecontrol group Aβ42 group, a large number of spots emerge, whichdemonstrates a strong T cell response. In the 25 μg protein+aluminiumadjuvant group, a large number of spots also emerge, which demonstratesthat the aluminium adjuvant could stimulate the body to produce acertain T cell response. Nevertheless, the 25 μgPP-10copy-Aβ1-6-loop2-S₁₄₉+CpG adjuvant group and 50 μg group have no orfewer positive spots, which demonstrates no evident T cell responseoccurring in the body, and as described in 3.1.1, this immunizationstrategy can stimulate the mice to produce the highest titer of specificantibodies against Aβ42. Considering the safety of vaccines, 25 gPP-10copy-Aβ1-6-loop2S₁₄₉+CpG adjuvant was selected as the optimalimmunization strategy.

3.2 Comparison of Immunological Effects of Three Recombinant P ParticleProtein Vaccines

After the experiments for determining immune dosages and immuneadjuvants of protein vaccines, the applicant first selected threerepresentative recombinant proteins, adopted the strategy of a proteinvaccine dosage of 25 μg/animal and CpG as the adjuvant, comparedimmunological effects of the three recombinant P particle proteinvaccines in female C57BL/6 mice aged 6-8 weeks by the two immunizationroutes via nasal and subcutaneous injections in order to compare theeffects of nasal and subcutaneous immunizations, and identify thedifferences in immunological effects of various proteins. Similar to themethod in 3.1.1, immunization was carried out every two weeks and fourtimes in total. Tails of the mice were cut and blood was taken beforeevery immunization. Serum was tested by ELISA detection. The mice weredivided into 7 test groups and 3 control groups. The immunogen andimmunization route for each group are shown as in FIG. 5.

TABLE 5 Immunization schemes of 3 representative P particle proteinsGroup Immunogen immunization route Control PBS subcutaneous group PBSnasal CpG subcutaneous CpG nasal test PP-1copy-Aβ1-6-loop2S₁₄₉ + CpGsubcutaneous group PP-10copy-Aβ1-6-loop2S₁₄₉ + CpG subcutaneousPP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉- subcutaneous3copy-Aβ1-6-loop3S₁₆₉ + CpG PP-1copy-Aβ1-6-loop2S₁₄₉ + CpG nasalPP-10copy-Aβ1-6-loop2S₁₄₉ + CpG nasalPP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉- nasal3copy-Aβ1-6-loop3S₁₆₉ + CpG

An ELISA plate was coated with Aβ1-42 as the antigen, using the methoddescribed in 3.1.1. A reaction was carried out using the immunized mouseserum as the primary antibody, HRP goat-anti-mouse antibody as thesecondary antibody, TMB as the substrate, and sulfuric acid as thetermination solution. After the termination of reaction, absorbance wasdetected at 450 nm using a microplate reader. The contents of antibodiesin the serum were compared based on the absorbance. Experiment wascarried out using PBS as the negative control, and the commercialantibody 6e10 (purchased from Covance Corporation) as the positivecontrol, and using the sera before and after immunizing mice with threeproteins as the test groups. Results are shown in FIG. 6. All the threeproteins can stimulate the mouse body to produce specific antibodiesagainst Aβ42, which demonstrates that the vaccines of the presentinvention have good immunogenicity. Standard curve was plotted using thecommercial antibody 6e10 as the positive control in order to calculatethe concentration of antibodies. The comparison results of immunologicaleffects are shown in FIG. 6. The two immunization routes of the threeproteins can all produce specific antibodies against Aβ42, the OD valuesof which are all significantly higher than those of the PBS group andCpG group. Moreover, with the increase of immunization times, theconcentration of the antibody continuously increases and can reach thehighest value at the fourth immunization. By comparison, it can be seenthat the subcutaneous immunization has better effect than the nasalimmunization.

3.3 Comparison of Immunological Effects of Various Recombinant PParticle Protein Vaccines

The applicant determined that the adopted immune dosage was 25 μg/animaland the immunization route was subcutaneous injection by the experimentsfor determining immune dosages and immune adjuvants of protein vaccines,and immunological experiments of three representative recombinantproteins, and determined the immunological effects of 127 candidatevaccines in female C57BL/6 mice aged 6-8 weeks. Immunization proceduresand method are as described in 3.2. Comparison results of immunologicaleffects are shown in Table 6.

TABLE 6 Concentrations of Aβ42 antibody produced by mice stimulated withprotein vaccines having 127 different forms of P particles as theimmunogens group Concentration of the produced Aβ42 number antibodyafter the fourth immunization 1 94.88 2 90.28 3 64.55 4 29.09 5 85.62 677.10 7 65.75 8 55.98 9 101.79 10 153.34 11 103.01 12 63.67 13 38.57 1440.87 15 48.89 16 34.82 17 89.98 28 111.76 19 77.09 20 38.78 21 105.8722 162.36 23 55.38 24 44.70 25 162.78 26 189.83 27 137.62 28 82.09 29187.23 30 83.42 31 56.67 32 54.31 33 87.47 34 77.21 35 68.47 36 42.12 3798.09 38 67.92 39 21.83 40 34.01 41 108.53 42 145.61 43 158.96 44 43.5745 76.37 46 64.89 47 56.17 48 29.02 49 68.78 50 75.26 51 77.15 52 39.5853 130.09 54 144.98 55 36.75 56 24.98 57 99.90 58 78.79 59 178.46 6071.14 61 88.82 62 22.19 63 109.70 64 142.06 65 20.08 66 19.98 67 111.4968 109.15 69 41.78 70 39.75 71 96.58 72 123.43 73 169.92 74 178.83 75120.47 76 21.10 77 102.80 78 197.09 79 95.03 80 76.78 81 55.87 82 23.5683 139.92 84 159.80 85 25.73 86 33.09 87 87.82 88 58.97 89 27.41 9024.46 91 73.45 92 82.67 93 168.21 94 116.81 95 82.39 96 43.08 97 142.9798 158.03 99 103.64 100 59.82 101 21.11 102 11.87 103 165.27 104 66.72105 47.86 106 34.56 107 150.98 108 75.73 109 99.17 110 45.43 111 31.25112 67.87 113 155.87 114 101.23 115 135.15 116 79.75 117 202.44 118215.93 119 245.12 120 222.76 121 23.89 122 149.78 123 152.29 124 166.09125 113.11 126 189.83 127 160.08

In the above P particles, the 17 P particles, PP-10copy-Aβ1-6-loop1G₇₂,PP-1copy-Aβ1-6-loop2S₁₄₉, PP-10copy-Aβ1-6-loop2S₁₄₉,PP-10copy-Aβ1-6-loop3G₁₆₉, PP-20copy-Aβ1-6-loop3G₁₆₉,PP-3copy-Aβ1-6-loop1G₇₂-3 copy-Aβ1-6-loop2S₁₄₉,PP-10copy-Aβ1-15-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉,PP-20copy-Aβ1-15-loop1G₇₂-40copy-Aβ1-6-loop2S₁₄₉,PP-3copy-Aβ1-12-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉, PP-1copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉,PP-20copy-Aβ1-12-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉,PP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-12-loop3G₁₆₉,PP-1copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop3G₁₆₉,PP-3copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-12-loop3G₁₆₉,PP-1copy-Aβ1-6-loop1G₇₂-1copy-Aβ1-6-loop2S₁₄₉-1copy-Aβ1-6-loop3 G₁₆₉,PP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉,PP-10copy-Aβ1-6-loop1G₇₂-10copy-Aβ1-6-loop2S₁₄₉-10copy-Aβ1-6-loop3G₁₆₉,have good immunological effects and can induce the production of highconcentrations of antibodies.

The standard curve is plotted in accordance with positive antibody 6e10,and its linear fitting curve is y=47.692x+0.2964, R2=0.99. According tothis standard curve, the concentration of the antibody produced byinduction can be calculated within the linear range, whereinPP-3copy-Aβ1-6-loop1G₇₂-3copy-Aβ1-6-loop2S₁₄₉-3copy-Aβ1-6-loop3G₁₆₉produce antibodies in a concentration of about 245.12 μg/mL after thefourth immunization. Therefore, the present invention has goodimmunological effects and induces a high concentration of Aβ1-42antibody in mouse serum after immunization. The present invention hasvery good therapeutical effects and is a protein vaccine with greatpotentials for treating AD.

The invention claimed is:
 1. A recombinant P particle formed from anorovirus capsid P protein chimerized with a Aβ1-m peptide, wherein m isan integer ranging from 6 to 15, and the recombinant P particle forms anordered and repetitive antigen array, and the amino acid sequence of atleast one of the Aβ1-m peptides is embedded into loop1, loop2 and/orloop3 of the norovirus capsid P protein, wherein the amino acid sequenceof the norovirus capsid P protein comprises the sequence of SEQ ID NO:1, and wherein N1 Aβ1-m peptide sequences are embedded behind one ormore amino acid sites selected from the group consisting of amino acids70-74 of SEQ ID NO: 1, i.e. 170, A71, G72, T73 and Q74; N2 Aβ1-m peptidesequences are embedded behind one or more amino acid sites selected fromthe group consisting of amino acids 148-151 of SEQ ID NO: 1, i.e. T148,S149, N150 and D151; and N3 Aβ1-m peptide sequences are embedded behindone or more amino acid sites selected from the group consisting of aminoacids 168-171 of SEQ ID NO: 1, i.e. D168, G169, S170 and T171; whereinN1, N2 and N3 each are independently selected from an integer rangingfrom 0-40, and N1+N2+N3≥1.
 2. The recombinant P particle according toclaim 1, wherein multiple consecutive Aβ1-m peptide sequences embeddedinto the norovirus capsid P protein are linked directly or via apolypeptide linker.
 3. The recombinant P particle according to claim 1,wherein the Aβ1-m peptide is linked to the norovirus capsid P proteindirectly or via a polypeptide linker.
 4. The recombinant P particleaccording to claim 1, wherein the Aβ1-m peptide sequence is an aminoacid sequence comprised in sequences selected from SEQ ID NOs: 2-8. 5.The recombinant P particle according to claim 1, wherein the chimerizednorovirus capsid P protein is encoded by nucleic acid sequences of SEQID NO: 15-SEQ ID NO:
 141. 6. A nucleic acid encoding the recombinant Pparticle according to claim 1, wherein the nucleic acid has sequences ofSEQ ID NO: 15-SEQ ID NO:
 141. 7. A pharmaceutical composition used forpreventing or treating Alzheimer's disease, comprising the recombinant Pparticle according to claim 1 and a pharmaceutically acceptable carrier.8. Use of the recombinant P particle according to claim 1 in themanufacture of a medicament for treating or preventing Alzheimer'sdisease, wherein the medicament is a vaccine.
 9. A method for preparingthe recombinant P particle according to claim 1, comprising thefollowing steps: i) obtaining an expression vector comprising a nucleicacid encoding a norovirus capsid P protein chimerized with a Aβ1-mpeptide, wherein m is an integer ranging from 6 to 15; ii) transferringthe expression vector into a receptor cell; iii) expressing thechimerized norovirus capsid P protein, and allowing it to self-assembleinto a recombinant P particle.